Composition containing Lactobacillus plantarum GKD7 for improving osteoarthritis and degenerative arthritis

Abstract

To provide a composition containing Lactobacillus plantarum GKD7 for improving osteoarthritis and degenerative joint disease.SOLUTION: This composition contains Lactobacillus plantarum GKD7 as an active ingredient, and is capable of alleviating joint pain caused by osteoarthritis, reducing the expression level of IL-1β in cartilage and / or synovial tissue, and improving osteoarthritis.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a probiotic-containing composition for improving musculoskeletal disorders, and in particular to a Lactobacillus plantarum GKD7-containing composition for improving osteoarthritis and degenerative arthritis. [Background technology]

[0002] Osteoarthritis (OA), also known as degenerative arthritis, is a chronic degenerative inflammatory disease occurring in joints. Osteoarthritis is usually accompanied by joint pain that is relieved after rest but worsens after activity. Then, symptoms of osteoarthritis further include joint ankylosis that improves after activity but recurs after rest. According to optical imaging analysis, damage associated with osteoarthritis often occurs in the synovial tissue, cartilage tissue and / or subchondral bone of the affected area of ​​osteoarthritis, such as joint space narrowing, osteophyte formation, subchondral bone sclerosis, subchondral bone cyst, bone damage, cartilage damage, inflammation of cartilage tissue and / or synovial tissue. Next, according to molecular biological research analysis, the levels of inflammatory cytokines are indeed higher in the serum, synovial tissue and cartilage tissue of patients with osteoarthritis compared with healthy general population.

[0003] The inflammatory cytokines may include, but are not limited to, interleukin (IL)-1β and tumor necrosis factor (TNF)-α. In osteoarthritis research, inflammatory cytokines play an important role in causing destructive changes in cartilage and structural changes in subchondral bone, so their reduction or inhibition is an important therapeutic goal. For example, inflammatory cytokines secreted from synovial fibroblasts and chondrocytes in osteoarthritis-affected areas promote the synthesis of proteolytic enzymes, which degrade the extracellular matrix of the joints, making osteoarthritis more severe.

[0004] Currently, the main treatment for osteoarthritis is to administer non-steroidal anti-inflammatory drugs (NSAIDs) or corticosteroids to reduce joint pain, swelling and stiffness caused by osteoarthritis. However, non-steroidal anti-inflammatory drugs or corticosteroids have undesirable side effects, some of which may even worsen osteoarthritis. When joint pain or dysfunction becomes too severe to be treated with drugs, patients may eventually have no choice but to consider artificial joint replacement.

[0005] Probiotics can promote the ecological balance of the host's microbiota and increase the number of live bacteria that bring health benefits to the host. Previous studies have demonstrated that some probiotics have anti-inflammatory effects. However, in order to maintain the growth activity of the live bacteria in probiotics, probiotics need to be produced and / or stored under specific conditions, and the dosage form and / or type of related products are limited.

[0006] Therefore, in order to solve the above problems, there is an urgent need for a killed bacteria-containing composition that improves osteoarthritis. Summary of the Invention

[0007] Therefore, one aspect of the present invention provides a Lactobacillus plantarum GKD7-containing composition for improving osteoarthritis, which contains killed Lactobacillus plantarum GKD7 as an active ingredient and relieves joint pain in a subject caused by osteoarthritis.

[0008] Another aspect of the present invention provides a Lactobacillus plantarum GKD7-containing oral composition for improving osteoarthritis, comprising killed Lactobacillus plantarum GKD7 as an active ingredient, and relieving joint pain in a subject caused by osteoarthritis.

[0009] According to the above aspect of the present invention, there is proposed a Lactobacillus plantarum GKD7-containing composition for improving osteoarthritis, which contains killed Lactobacillus plantarum GKD7 as an active ingredient, and the Lactobacillus plantarum GKD7 was deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (NITE) of Japan on November 6, 2020, and has the accession number NITE BP-03313.

[0010] According to some embodiments of the present invention, killed Lactobacillus plantarum GKD7 is obtained after Lactobacillus plantarum GKD7 is subjected to a fermentation process and an inactivation process. In some embodiments, the fermentation process is carried out with a fermentation substrate in an anaerobic atmosphere at 25°C to 40°C for 16 hours to 24 hours.

[0011] According to some embodiments of the present invention, the deactivation step includes a thermal deactivation step, a chemical deactivation step and / or a radiation deactivation step.

[0012] According to some embodiments of the present invention, when the subject is a rat, the effective dosage is 10 mg / kg body weight / day to 1000 mg / kg body weight / day.

[0013] According to some embodiments of the present invention, when the subject is a human, the effective dosage is between 1 mg / kg body weight / day and 165 mg / kg body weight / day.

[0014] According to another aspect of the present invention, there is proposed a Lactobacillus plantarum GKD7-containing oral composition for improving osteoarthritis, which comprises killed Lactobacillus plantarum GKD7 bacteria as an active ingredient, and relieves joint pain in subjects, and Lactobacillus plantarum GKD7 has the accession number NITE BP-03313.

[0015] According to some embodiments of the present invention, subjects administered the oral composition exhibit less side-to-side weight bearing compared to control subjects not administered the composition.

[0016] According to some embodiments of the present invention, subjects administered the oral composition exhibit reduced expression of IL-1β in cartilage and / or synovial tissue compared to control subjects not administered the composition.

[0017] According to some embodiments of the invention, the composition is administered to the subject three to seven times per week.

[0018] According to some embodiments of the present invention, the composition is administered to the subject for a period of 2 to 8 weeks. Effect of the Invention

[0019] By applying a Lactobacillus plantarum GKD7-containing composition for improving osteoarthritis and degenerative arthritis, which contains killed Lactobacillus plantarum GKD7 cells according to the present invention as an active ingredient, joint pain can be alleviated and the expression level of IL-1β in cartilage tissue and / or synovial tissue can be reduced, thereby improving osteoarthritis. The following detailed description of the accompanying drawings will make the above and other objects, features, advantages and embodiments of the present disclosure more apparent and understandable. [Brief description of the drawings]

[0020] [Figure 1] 1 is a graph showing the change in load difference 6 weeks after surgery in different groups of rats according to one embodiment of the present invention. [Diagram 2] 1 is a graph showing the change in load difference 6 weeks after surgery in different groups of rats according to one embodiment of the present invention. [Diagram 3] 3A-3G are box plots showing bone mineral density, bone content, bone volume fraction, bone areal density, trabecular thickness, trabecular number and trabecular spacing of different groups, respectively, according to one embodiment of the present invention. [Figure 4]4A to 4C respectively show the OARSI score, the cartilage degeneration score and the synovial inflammation score of different groups according to one embodiment of the present invention. [Diagram 5] 5A and 5B are box plots showing the IHC scores of IL-1β and the IHC scores of TNF-α in cartilage tissues of different groups according to one embodiment of the present invention. [Figure 6] 6A and 6B are box plots showing the IHC scores of IL-1β and the IHC scores of TNF-α in synovial tissues of different groups according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] In the event that a definition or use of a term in a reference contradicts or is contrary to a definition of that term in this specification, the definition of that term in this specification shall control but not in the reference. Also, unless otherwise defined by context, singular terms may include plurals and plural terms may include the singular.

[0022] As described above, the present invention provides a Lactobacillus plantarum-containing composition for improving osteoarthritis, which contains killed Lactobacillus plantarum GKD7 as an active ingredient. The Lactobacillus plantarum GKD7 was deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (NITE) of Japan on November 6, 2020 (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, Postal Code: 292-0818), and has the accession number NITE BP-03313. This strain was also deposited at the Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI), Taiwan, No. 331 Food Road, Hsinchu City, Hsinchu, 300193, on March 6, 2019, with the accession number BCRC 910877.

[0023] The "killed bacteria" of Lactobacillus plantarum GKD7 described herein refers to Lactobacillus plantarum GKD7 cells that have lost growth activity. In some embodiments, the killed bacteria of Lactobacillus plantarum GKD7 are obtained after Lactobacillus plantarum GKD7 undergoes a fermentation process and an inactivation process. In some embodiments, the fermentation process is carried out at 25°C to 40°C, or 35°C to 40°C, or 37°C so that Lactobacillus plantarum GKD7 has a favorable growth activity and obtains a large number of Lactobacillus plantarum GKD7 cells. In some embodiments, the fermentation process is carried out for 16 hours to 24 hours, or 18 hours so that the growth curve of Lactobacillus plantarum GKD7 is between the logarithmic phase and the lag phase. In some embodiments, the fermentation process is carried out in an anaerobic atmosphere. The air composition in the anaerobic atmosphere is not particularly limited, but the oxygen content must be 0. In one embodiment, the anaerobic atmosphere may include, but is not limited to, 75%-85% nitrogen gas, 5%-15% hydrogen gas, and 5%-15% carbon dioxide. It should be noted that the active substances of the killed bacteria may include, but are not limited to, lipids, sugars, excess water proteins, glycoproteins, glycolipids, and / or other biomolecules in the cell wall, cytoplasm, and / or metabolites, and that it is not reasonably expected that the effect of improving osteoarthritis and osteoarthritis can be achieved by utilizing a single substance therein and / or excluding unknown components that cannot be separated. Therefore, the killed bacteria of Lactobacillus plantarum GKD7 according to the present invention cannot be defined by components, characteristics, or parameters, i.e., the present invention may not be possible or practical to define by components, characteristics, or parameters.

[0024] The fermentation substrate for the fermentation step may be, but is not limited to, a commercially available fermentation substrate such as MRS broth. In some embodiments, the fermentation substrate may include, but is not limited to, 1 wt% to 10 wt% sugars, 0.1 wt% to 5.0 wt% yeast extract, 0.1 wt% to 5.0 wt% peptone, 0.01 wt% to 2.00 wt% trace elements, 0.01 wt% to 0.10 wt% cysteine, 0.05 wt% to 1.00 wt% TWEEN-80, and the remaining amount of water. The sugars may include, but are not limited to, monosaccharides and / or disaccharides. In some embodiments, the sugars may be selected from the group consisting of glucose, galactose, fructose, maltose, lactose, sucrose, and combinations thereof. The trace elements may be, for example, selected from the group consisting of magnesium ions, potassium ions, sulfate ions, hydrogen phosphite ions, and combinations thereof.

[0025] In some embodiments, after the fermentation step, a solid-liquid separation step may be optionally performed to remove the fermentation substrate. The solid-liquid separation step is not particularly limited, but may be performed to remove the fermentation substrate. In some embodiments, the solid-liquid separation step may include, but is not limited to, a filtration step and / or a centrifugation step.

[0026] The method of the inactivation step is not particularly limited, but it is sufficient that Lactobacillus plantarum GKD7 loses growth activity. In some embodiments, the inactivation step may include, but is not limited to, a heat inactivation step, a chemical inactivation step, and / or a radiation inactivation step. In some embodiments, the heat inactivation step is performed at 110°C to 120°C under saturated vapor pressure for 10 minutes to 20 minutes. It should be noted that experiments have demonstrated that killed Lactobacillus plantarum GKD7 loses growth activity but has osteoarthritis improving activity.

[0027] In some embodiments, the killed Lactobacillus plantarum GKD7 may be further dried to obtain a freeze-dried powder and extend its shelf life. In some embodiments, the freeze-dried powder contains Lactobacillus plantarum GKD7 at a concentration of 5.5×10 7 Equivalent to CFU / g.

[0028] Killed Lactobacillus plantarum GKD7 can improve osteoarthritis. Osteoarthritis, as described herein, is a degenerative joint disease that causes destruction of articular cartilage and subchondral bone, resulting in joint pain and further altering the structure of cartilage and / or bone. "Improving osteoarthritis" as described herein means alleviating joint pain, reducing damage to cartilage and bone, and / or reducing the level of inflammatory cytokines. In some embodiments, the evaluation items for evaluating whether osteoarthritis has been improved may include, but are not limited to, the degree of joint pain, the expression level of inflammatory cytokines in cartilage tissue and synovial tissue, the degree of bone damage, and / or the degree of cartilage damage. In some embodiments, the degree of joint pain may be evaluated by load difference, since osteoarthritis is generally not symmetrical. The smaller the load difference, the lower the degree of pain in the affected area of ​​osteoarthritis. In some embodiments, the inflammatory cytokines may include, but are not limited to, Interleukin (IL)-1β and Tumor necrosis factor (TNF)-α.

[0029] In some embodiments, the degree of bone damage may be assessed by bone mineral density (BMD), bone mineral content (BMC), bone volume fraction, bone area fraction, trabecular thickness, trabecular number, and trabecular separation. In some embodiments, the degree of cartilage damage may be scored by the histological scoring system of rat joints recommended by the Osteoarthritis Research Society International (OARSI).

[0030] Experiments have demonstrated that live and / or killed Lactobacillus plantarum GKD7 can improve osteoarthritis by oral administration. Specifically, compared with control subjects not administered Lactobacillus plantarum GKD7, subjects administered Lactobacillus plantarum GKD7 have lower pain levels, lower bone damage levels, lower cartilage damage levels, and lower levels of inflammatory cytokines in cartilage tissue and synovial tissue. Therefore, Lactobacillus plantarum GKD7 can be applied to compositions for improving osteoarthritis and osteoarthritis. In the above examples, the composition may contain an effective dose of killed Lactobacillus plantarum GKD7 as an active ingredient, but is not limited thereto.

[0031] The test subject may be a mammalian animal, such as, but not limited to, a rat, a mouse, a rabbit, a cat, a dog, or a human. In some embodiments, when the test subject is a rat, the effective dosage of Lactobacillus plantarum GKD7 may be, for example, 10 mg / kg body weight / day to 1000 mg / kg body weight / day, and a relatively small dosage, such as 10 mg / kg to 500 mg / kg or 10 mg / kg to 100 mg / kg, may achieve a favorable effect. In some embodiments, when the test subject is a human, the effective dosage of Lactobacillus plantarum GKD7 may be, for example, 1 mg / kg body weight / day to 165 mg / kg body weight / day, such as 1 mg / kg body weight / day to 100 mg / kg body weight / day, or 1 mg / kg body weight / day to 50 mg / kg body weight / day. Animal experiments have shown that administering 100mg / kg-1000mg / kg of Lactobacillus plantarum GKD7 can indeed improve osteoarthritis, but it should be noted that in this animal experiment, osteoarthritis was induced by previous cruciate ligament transection surgery, so the course of osteoarthritis is very fast, and in clinical applications, the same effect can be achieved with a relatively small dose. In some embodiments, when the examinee is a human, the effective dose of Lactobacillus plantarum GKD7 is 1mg / kg body weight / day-10mg / kg body weight / day. In the above embodiments, the examinee is either trying to prevent osteoarthritis or suffering from early osteoarthritis (at least one but less than four joint inflammation, and within two years of onset).

[0032] The Lactobacillus plantarum GKD7 should be administered to the examinee continuously for a certain period of time. In some embodiments, the Lactobacillus plantarum GKD7 may be administered to the examinee three to seven times a week, for example, five times a week. In some embodiments, the Lactobacillus plantarum GKD7 may be administered to the examinee for two to eight weeks, for example, or longer or shorter.

[0033] In some applications, the composition may be, for example, an oral composition. In some embodiments, the composition may be, for example, a pharmaceutical composition or a food composition. In one embodiment, the oral composition may optionally include a food- or medically acceptable carrier, excipient, diluent, adjuvant, preservative, filler, and / or additive.

[0034] The following describes the application of the present invention using several examples, but these are not intended to limit the present invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. EXAMPLES

[0035] 1. Preparation Example 1 1.1 Origin of the strain Lactobacillus plantarum GKD7 (hereinafter referred to as strain GKD7) was deposited at NITE on November 6, 2020, with accession number NITE BP-03313, and at BCRC on March 6, 2019, with accession number BCRC 910877. For the microbiological characteristics and culture method of strain GKD7, refer to the case of Taiwan Patent TW I733207B, which is also cited herein as a reference.

[0036] 1.2 Preparation of lyophilized powder A bacterial solution of the GKD7 strain was applied to an MRS medium and cultured in an anaerobic atmosphere (80% N2, 10% H2, and 10% CO2) at 37°C for 18 hours to obtain a single colony. The single colony was then inoculated into a culture solution, and a fermentation process was carried out in an anaerobic atmosphere at 37°C for another 18 hours to obtain a liquid culture. The culture solution contained 1% to 10% glucose, 0.1% to 5% yeast extract, 0.1% to 0.5% peptone, 0.01% to 2.00% trace elements, 0.01% to 0.10% cysteine, 0.05% to 1.00% TWEEN80, and the remaining amount of water, and the trace elements were selected from the group consisting of magnesium ions, potassium ions, sulfate ions, hydrogen phosphite ions, and combinations thereof. It should be noted that the type of culture medium does not affect the effect of the GKD7 strain in improving osteoarthritis, and the culture media listed above are not intended to limit the present invention.

[0037] Next, 15 wt% skim milk powder was added to the liquid culture, and a centrifugation step was performed at 1000 rpm to 15000 rpm to obtain live bacteria of the strain GKD7. Mixing the liquid culture with 15 wt% skim milk powder before the centrifugation step helps the bacteria to aggregate into balls, and increases the recovery rate of the bacteria. Then, the bacteria were mixed with 6 wt% to 50 wt% skim milk powder, and then a freeze-drying step was performed to obtain a freeze-dried powder of the live bacteria of the strain GKD7. It should be noted that the freeze-drying is merely for the purpose of facilitating administration, quantification, and storage, and is not intended to limit the present invention.

[0038] 2. Preparation Example 2 The liquid culture was subjected to a heat inactivation step at saturated vapor pressure of 110°C to obtain a heat inactivated liquid culture. The heat inactivated liquid culture was then subjected to a centrifugation step to obtain killed cells of the GKD7 strain. The killed cells of the GKD7 strain were then subjected to a freeze-drying step to obtain a freeze-dried powder of the GKD7 strain.

[0039] 3. Evaluation method and results Animal models Eight-week-old male Sprague Dawley (SD) rats were housed in stainless steel rat cages, and the temperature and humidity of the environment were 22±2°C, 60%-80%, and the light and dark periods were 12 hours each. During the test, the rats had free access to food and distilled water. The rats weighed 300g-350g and had matured skeletal development.

[0040] The rats were divided into a sham-operated control group (n=6), a surgical control group (n=6) and an experimental group 1 (n=6). The right hind limbs of the rats in the sham-operated control group, the surgical control group and the experimental group 1 were subjected to corresponding surgeries, the right hind limbs of the rats in the surgical control group and the experimental group 1 were subjected to anterior cruciate ligament transect (ACLT) including arthrotomy and anterior cruciate ligament fiber transection, and osteoarthritis was induced in the right hind limbs of the rats by ACLT, and the right hind limbs of the rats in the sham-operated control group were subjected to arthrotomy but their anterior cruciate ligament fibers were not transected.

[0041] Two days after surgery, 1 mL of distilled water was added to the diet of each group. Rats in experimental group 1 were given 100 mg / kg bw / day (5 × 10 11 To obtain a freeze-dried powder of live bacteria of the GKD7 strain (equivalent to CFU / g, which is equivalent to a human dose of 16.1 mg / kg body weight / day), the live freeze-dried powder of the GKD7 strain was added to the distilled water of experimental group 1 five days a week (Preparation Example 1).

[0042] Then, different doses of live and killed freeze-dried powders of GKD7 strain were tested. Eight-week-old rats were divided into six groups, sham-operated control group 2, surgical control group 2, experimental group 2, experimental group 3, experimental group 4, and positive control group. There were eight animals tested per group. The right hind limbs of rats in sham-operated control group 2 underwent arthrotomy, but their anterior cruciate ligament fibers were not severed. The right hind limbs of rats in surgical control group 2, experimental group 2, experimental group 3, experimental group 4, and positive control group underwent anterior cruciate ligament transection surgery.

[0043] After the rats had recovered for 1-2 days, 1 mL of distilled water was added to the feed of each group, 5 days per week, and the corresponding doses of live freeze-dried powder of GKD7 and dead freeze-dried powder of GKD7 were added to the distilled water of the different groups. This allowed the rats in experimental group 2 to obtain a low dose (100 mg / kg bw / day, the same as the dose in experimental group 1) of live freeze-dried powder of the GKD7 strain (Preparation Example 1), the rats in experimental group 3 to obtain a high dose (1 g / kg bw / day, equivalent to a human dose of 161.3 mg / kg body weight / day) of live freeze-dried powder of the GKD7 strain (Preparation Example 1), and the rats in experimental group 4 to obtain 1 g / kg bw / day of dead freeze-dried powder of the GKD7 strain (Preparation Example 2). Celecoxib (50 mg / kg) was added to the distilled water in the positive control group. Celecoxib is a commercially available analgesic that acts to inhibit the activity of cyclooxygenase (COX), thereby suppressing the synthesis of prostaglandin E2 (PGE2), thereby reducing inflammatory pain. 3.2 Static weight bearing test (also called incapacitance test)

[0044] Before surgery, the left and right hindlimbs of rats in the sham-operated control group, the operated control group, and the experimental group 1 were placed on separate sensor plates for 10 seconds, and the load difference between the left and right hindlimbs (unit: basis weight, normalized by the rat's body weight) was measured when the rat stood on its hindlimbs. After surgery, the above measurements were performed weekly. Generally, normal rats distribute their body weight equally between the left and right hindlimbs, so the larger the load difference between the left and right hindlimbs, the higher the degree of pain in the rat's right hindlimbs. The results of different groups were compared by paired t-test.

[0045] FIG. 1 is a graph showing the change in load difference 6 weeks after surgery in different groups of rats according to one embodiment of the present invention, where the horizontal axis represents time (unit: weeks) and the vertical axis represents load difference (unit: g), curves 110, 120 and 130 represent the sham surgery control group, the surgery control group and experimental group 1, respectively, with the symbol "*" indicating a statistically significant difference (p<0.05) from the sham surgery control group and the symbol "#" indicating a statistically significant difference (p<0.05) from the surgery control group.

[0046] As shown in FIG. 1, the load difference of the surgical control group (curve 120) was significantly higher than that of the sham-surgery control group (curve 110), and the load difference increased over time, which means that ACLT indeed induces osteoarthritis and causes joint pain. Conversely, the load difference of experimental group 1 significantly decreased 2 weeks after surgery, and 6 weeks after surgery, the load difference of experimental group 1 was only about 50% of that of the surgical control group. This result showed that the live freeze-dried powder of strain GKD7 can significantly alleviate joint pain caused by osteoarthritis after being administered for 14 days (2 weeks). It should be noted that there was no statistically significant difference (not shown) in the weight of the rats between each group, as shown by the weight measurements before and 6 weeks after surgery, indicating that ACLT does not affect the weight of the rats.

[0047] The sham control group 2, the operation control group 2, the experimental group 2, the experimental group 3, the experimental group 4 and the positive control group were also subjected to the static load test, the method of which has been described above and will not be repeated here.

[0048] FIG. 2 is a graph showing the change in load difference 6 weeks after surgery in different groups of rats according to one embodiment of the present invention, where the horizontal axis represents time (unit: weeks) and the vertical axis represents load difference (unit: g), with curves 210, 220, 230, 240, 250 and 260 representing sham surgery control group 2, surgery control group 2, experimental group 2, experimental group 3, experimental group 4 and the positive control group, respectively, with the symbol "#" indicating that there is a statistically significant difference from sham surgery control group 2 (curve 210) and the symbol "*" indicating that there is a statistically significant difference from surgery control group 2 (curve 220).

[0049] As shown in FIG. 2, compared with the surgical control group 2 (curve 220), the load difference was small for experimental group 2 (curve 230), experimental group 3 (curve 240), experimental group 4 (curve 250) and the positive control group (curve 260), indicating that the live freeze-dried powder of GKD7 and the killed freeze-dried powder of GKD7 can indeed relieve joint pain caused by osteoarthritis. Curve 240 representing experimental group 3 (high dose live freeze-dried powder of GKD7) and curve 260 representing the positive control group almost overlapped 4 to 6 weeks after surgery, indicating that the joint pain relieving effect of the high dose live freeze-dried powder of GKD7 is equivalent to that of a commercially available analgesic drug (celecoxib). Next, the curve 230 representing experimental group 2 (low dose live freeze-dried powder of GKD7 strain) and the curve 250 representing experimental group 4 (killed freeze-dried powder of GKD7 strain) had the same tendency between 1 week and 6 weeks after surgery, indicating that the live freeze-dried powder of GKD7 strain and the killed freeze-dried powder can have the same joint pain relieving effect. Therefore, the subsequent evaluation is to analyze the effect of low dose live freeze-dried powder of GKD7 strain on the rats of Example 1, but a person skilled in the art would understand that the same effect should be achieved by administering killed freeze-dried powder of GKD7 strain.

[0050] 3.3 Bone image analysis Six weeks after the ACLT operation, the rats in the sham operation control group, operation control group and experimental group 1 were sacrificed, and the bone parameters of the knee joint of the right hind leg of the rats were analyzed by micro-computerized tomography (Micro-CT, μ-CT), including bone mineral density, bone content, bone volume fraction, bone areal density, trabecular thickness, trabecular number and trabecular space. The micro-computerized tomography showed that the rats in the operation control group indeed had bone damage, including bone loss of the subchondral bone (not shown), but the degree of bone loss in the rats in experimental group 1 was lower than that in the operation control group (not shown).

[0051] 3A-3G are box plots showing bone mineral density (FIG. 3A), bone content (FIG. 3B), bone volume fraction (FIG. 3C), bone areal density (FIG. 3D), trabecular thickness (FIG. 3E), trabecular number (FIG. 3F), and trabecular space (FIG. 3G) of different groups according to one embodiment of the present invention, with the horizontal axis representing the groups, from left to right being the sham-operated control group, the operated control group, and the experimental group 1, respectively, and the vertical axis representing bone mineral density (unit: g / cm 3 ), bone content (unit: mg), bone volume fraction (i.e., the ratio of bone volume to total volume, abbreviated as BV / TV, unit: %), bone areal density (i.e., the ratio of bone surface area to total volume, abbreviated as BS / TV, unit: 1 / mm), trabecular thickness (abbreviated as Tb.Th, unit: mm), trabecular number (abbreviated as Tb.N, unit: 1 / mm), and trabecular space (abbreviated as Tb.Sp, unit: mm), where the symbol "*" indicates a statistically significant difference (p<0.05) from the sham-operated control group, and the symbol "#" indicates a statistically significant difference (p<0.05) from the operated control group.

[0052] As shown in Figures 3A-3G, compared with the sham-operated control group, the operation control group had significantly decreased bone mineral density, bone content, bone volume fraction, bone areal density, trabecular thickness and trabecular number in the knee joint of the right hind leg of rats, but significantly increased trabecular space, indicating that the induction of osteoarthritis by ACLT can indeed cause bone damage such as bone loss and bone destruction. Conversely, the above bone parameters of the knee joint of the right hind leg of rats in experimental group 1 were significantly improved, indicating that the live freeze-dried powder of the GKD7 strain can indeed ameliorate bone damage caused by osteoarthritis. 3.4 Histological analysis

[0053] After analyzing the bone images, the knee joint tissues of the right hind limbs of the rats were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 37°C for 24 hours, and then decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 2 weeks. After dehydration with increasing concentrations of ethanol, the knee joint tissues were embedded in paraffin blocks and cut into tissue sections with a thickness of 5 μm. The tissue sections were then stained with hematoxylin and eosin (H&E) and Safranin-O / Fast Green, and the histopathological changes of the knee joint tissues were observed under an optical microscope. As can be seen from the tissue sections, the knee joint tissue of the rats in the surgical control group indeed showed pathological changes in the cartilage tissue and cartilage damage such as hyperplasia of the synovial tissue (not shown), but the degree of cartilage damage in the rats in experimental group 1 was less than that of the rats in the surgical control group (not shown).

[0054] The degree of cartilage damage in knee joint tissue was quantified using the rat joint histological scoring system recommended by Osteoarthritis Research Society International (OARSI) score, cartilage degeneration score and synovial inflammation score. The Osteoarthritis Research Society International score, cartilage degeneration score and synovial inflammation score are well known to those skilled in the art and will not be described again here.

[0055] Figures 4A to 4C each show the International Osteoarthritis Society score, cartilage degeneration score, and synovial inflammation score of different groups according to one embodiment of the present invention, where the horizontal axis represents the groups, from left to right being the sham surgery control group, the surgery control group, and experimental group 1, respectively, and the vertical axis represents the International Osteoarthritis Society score, cartilage degeneration score, and synovial inflammation score (unit: points), respectively, with the symbol "*" indicating a statistically significant difference (p<0.05) from the sham surgery control group, and the symbol "#" indicating a statistically significant difference (p<0.05) from the surgery control group.

[0056] As shown in Figures 4A-4C, the International Osteoarthritis Society score, cartilage degeneration score, and synovial inflammation score of the surgical control group were significantly improved compared to the sham-operated control group, indicating that osteoarthritis induced by ACLT can indeed cause cartilage damage. And, compared to the surgical control group, the International Osteoarthritis Society score, cartilage degeneration score, and synovial inflammation score of experimental group 1 were significantly decreased, indicating that the live freeze-dried powder of GKD7 strain can indeed improve cartilage damage caused by osteoarthritis.

[0057] 3.5 Immunohistochemistry (IHC) The tissue sections were immersed in a 3% hydrogen peroxide solution to suppress endogenous catalase activity, and then in a PBS solution containing 3% bovine serum albumin to reduce the occurrence of nonspecific background. After primary antibody incubation with anti-IL-1β or anti-TNF-α antibodies at 4°C, the tissue sections were incubated with secondary antibodies at room temperature for 1 hour, and then stained with diaminobenzidine (DAB). The tissue sections were observed under an optical microscope. The IHC scores represent the immunoreactive signals of IL-1β and TNF-α, and the higher the IHC score, the stronger the immunoreactive signal, i.e., the higher the expression level of IL-1β or TNF-α.

[0058] 5A-5B are box plots showing the IHC scores of IL-1β (FIG. 5A) and TNF-α (FIG. 5B) in cartilage tissues of different groups according to one embodiment of the present invention, with the horizontal axis representing the groups, from left to right being the sham-operated control group, the surgical control group, and experimental group 1, respectively, and the vertical axis representing the IHC scores of IL-1β and TNF-α (unit: points). As shown in FIG. 5A and FIG. 5B, compared with the sham-operated control group, the surgical control group had significantly increased IHC scores of IL-1β and TNF-α, indicating high expression of inflammatory cytokines such as IL-1β and TNF-α in cartilage tissues of osteoarthritis induced by ACLT. Conversely, compared with the surgical control group, the IHC scores of IL-1β and TNF-α in experimental group 1 were significantly decreased, indicating that the live lyophilized powder of GKD7 strain could indeed reduce the expression levels of inflammatory cytokines in osteoarthritis cartilage tissue.

[0059] 6A-6B are box plots showing the IHC scores of IL-1β (FIG. 6A) and TNF-α (FIG. 6B) of synovial tissues of different groups according to one embodiment of the present invention, with the horizontal axis representing the groups, from left to right, the sham-operated control group, the surgical control group, and the experimental group 1, respectively, and the vertical axis representing the IHC scores of IL-1β and TNF-α (unit: points). As shown in FIG. 6A and FIG. 6B, compared with the sham-operated control group, the surgical control group had significantly increased IHC scores of IL-1β and TNF-α, indicating that the expression of inflammatory cytokines in the synovial tissues of osteoarthritis induced by ACLT was indeed high. Conversely, compared with the surgical control group, the experimental group 1 had significantly decreased IHC scores of IL-1β and TNF-α, indicating that the live freeze-dried powder of the GKD7 strain can indeed reduce the expression of inflammatory cytokines in the synovial tissues of osteoarthritis.

[0060] In summary, the present invention has been described by exemplifying a Lactobacillus plantarum GKD7-containing composition for simply improving osteoarthritis and osteoarthritis by a specific dosage amount, a specific dosage form, a specific detection method, or a specific evaluation method. However, for those skilled in the art, other dosage amounts, other dosage forms, other detection methods, or other evaluation methods may also be applied to the composition for improving osteoarthritis and osteoarthritis without departing from the spirit and scope of the present invention, and are not limited to the above. For example, the composition may be, for example, a pharmaceutical composition or a food composition, and may selectively contain medically and food-acceptable carriers, excipients, and / or additives, and may be in the form of a powder, tablet, granule, suppository, microcapsule, ampoule, liquid spray, or embolization agent. In another example, Lactobacillus plantarum GKD7 may be cultured using other suitable media or other culture environments, and the obtained bacteria should also have the above-mentioned improving effect on osteoarthritis. In other embodiments, effects achievable with a low dose of live bacteria (Experimental Group 1) include improving bone damage, cartilage damage, inflammation of cartilage tissue and / or synovial tissue due to osteoarthritis, etc., which can also be achieved with a high dose of live bacteria (Experimental Group 2) and a high dose of killed bacteria (Experimental Group 4).

[0061] According to the above examples, the Lactobacillus plantarum GKD7-containing composition for improving osteoarthritis and osteoarthritis according to the present invention can not only relieve joint pain caused by osteoarthritis by using killed Lactobacillus plantarum GKD7 bacteria, but also improve bone damage, cartilage damage, and inflammation of cartilage tissue and / or synovial tissue caused by osteoarthritis, and has the advantage that it can be used as an active ingredient in the preparation of various compositions and applied to the improvement of osteoarthritis in the future.

[0062] While the present invention has been disclosed above in terms of several specific embodiments, other embodiments are possible, and therefore, the spirit and scope of the invention as defined by the appended claims should not be limited to the embodiments described herein. [Explanation of symbols]

[0063] Curves 110, 120, 130, 210, 220, 230, 240, 250, 260

Claims

1. A composition for improving osteoarthritis, comprising an effective dose of killed Lactobacillus plantarum GKD7 as an active ingredient, wherein the composition relieves joint pain in an examinee caused by osteoarthritis after administration to the examinee for two weeks, the Lactobacillus plantarum GKD7 was deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (NITE) on November 6, 2020, with the accession number NITE BP-03313, and the killed Lactobacillus plantarum GKD7 is a heat-inactivated cell.

2. 10. The composition of claim 1, wherein the effective dosage is 10 mg / kg body weight / day to 1000 mg / kg body weight / day when the subject is a rat.

3. 10. The composition of claim 1, wherein the effective dosage is from 1 mg / kg body weight / day to 165 mg / kg body weight / day when the subject is a human.

4. An oral composition for improving osteoarthritis, comprising killed Lactobacillus plantarum GKD7 as an active ingredient, the composition relieving joint pain in an examinee after administration to the examinee for two weeks, the Lactobacillus plantarum GKD7 having an accession number of NITE BP-03313, and the killed Lactobacillus plantarum GKD7 cells being heat-inactivated cells.

5. The oral composition according to claim 4 , wherein the test subjects administered the oral composition have a smaller difference in weight load between the left and right sides compared to control test subjects not administered the oral composition.

6. The oral composition of claim 4, wherein the expression level of IL-1β in cartilage tissue and / or synovial tissue is reduced in the subject administered the oral composition compared to a control subject not administered the oral composition.

7. 5. The oral composition of claim 4, wherein the oral composition is administered to the test subject 3 to 7 times per week.

8. The oral composition of claim 4, wherein the oral composition is administered to the test subject for a period of 2 to 8 weeks.

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